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An Archaeal Iron-Oxidizing Extreme Acidophile Important in Acid Mine Drainage

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Science  10 Mar 2000:
Vol. 287, Issue 5459, pp. 1796-1799
DOI: 10.1126/science.287.5459.1796

Abstract

A new species of Archaea grows at pH ∼0.5 and ∼40°C in slime streamers and attached to pyrite surfaces at a sulfide ore body, Iron Mountain, California. This iron-oxidizing Archaeon is capable of growth at pH 0. This species represents a dominant prokaryote in the environment studied (slimes and sediments) and constituted up to 85% of the microbial community when solution concentrations were high (conductivity of 100 to 160 millisiemens per centimeter). The presence of this and other closely relatedThermoplasmales suggests that these acidophiles are important contributors to acid mine drainage and may substantially impact iron and sulfur cycles.

The oxidative dissolution of metal sulfide minerals causes the formation of acid mine drainage (AMD) and plays an important role in the geochemical sulfur cycle. Sulfides (primarily pyrite, FeS2) that are exposed to air and water through geological or mining activities undergo oxidative dissolution and generate sulfuric acid by the reaction FeS2 + 14Fe3+ + 8H2O → 15Fe2+ + 2SO4 2− + 16H+ (1). Mining and extraction mobilizes ∼150 × 1012 g of sulfur per year, contributing ∼50% to the net river transport of sulfate to the ocean, which is about half of the sulfate input into the ocean (2).

Microorganisms accelerate the rate of pyrite dissolution through regeneration of Fe3+ (1, 3), the primary pyrite oxidant at low pH (4–6). At Iron Mountain, an AMD site in northern California, the iron-oxidizing bacterium Thiobacillus ferrooxidans, previously thought to be the most important iron-oxidizing species, played a minor role in pyrite oxidation (7, 8). Instead, Archaea constituted a large proportion (>50%) of the prokaryote population at important sites of acid generation during the dry summer and fall months (8). Here we describe the isolation, identification, and physiology of an Archaeon from Iron Mountain and the ecological niche it, and closely related Archaea, occupy.

We started enrichment cultures with sediments and mine water collected in July 1997 from the B-drift at the Richmond 5-way. The 5-way is a site ∼500 m into the mountain, situated within the remaining pyrite ore-body at a junction between five tunnels. Temperatures at the Richmond 5-way are ∼40°C, and the pH of the waters is 0 to 1 (8). The low-pH conditions at Iron Mountain support extremely high metal concentrations in solutions; iron has been measured as high as 111 g liter−1, and copper, arsenic, cadmium, and zinc all have been measured in the tens to tenths of grams per liter range (9). Specific conductance at the time of collection was ∼120 mS cm−1 (8). We grew enrichment cultures at 37°C in pH 1 medium (10), using pyrite sediments as the energy source. We performed DNA extractions and constructed clone libraries for two of the enrichment cultures and sequenced representative clones (11). A high proportion of the clones formed a monophyletic cluster withFerroplasma acidiphilum (12), an iron-oxidizing chemolithotrophic, autotrophic Archaeon within the orderThermoplasmales (Fig. 1). TheThermoplasmales are acidophilic Archaea that possess only a single, peripheral (cytoplasmic) membrane (12). Subsequently, from an enrichment culture, an isolate (fer1; Fig. 2) was obtained by serial dilution in pH 1.5 medium (10) that was supplemented with 20 g liter−1 FeSO4·7H2O and 0.02% yeast extract. Fluorescent in situ hybridizations [FISH (13)] with a probe designed for the Ferroplasmacluster FER656 (13) confirmed that the isolate was aFerroplasma species.

Figure 1

Phylogenetic relationship of clone ECB1 within the Archaeal domain based on comparison of partial 16S ribosomal DNA sequences. Clone sequences were aligned to representative sequences from the Ribosomal Database Project [RDP (25)] and GenBank (26) databases. Phylogenetic analysis was performed with the ARB software package (27). Sequences were aligned and reduced to 819 comparable positions with a filter created in ARB. A dendogram was constructed in ARB by evolutionary distance (neighbor joining) and compared to inference by maximum likelihood [fastDNAml; RDP (25)]. Accession numbers are shown for the comparison sequences that were obtained from the RDP (25). Branch points on the dendogram are indicated by filled circles when supported by both evolutionary distance and maximum likelihood analyses.

Figure 2

Scanning (SEM) and transmission (TEM) electron micrographs of F. acidarmanus isolate fer1. (A) Cryo-SEM (bar, 500 nm). Fer1 cells in late log growth were prepared for high-pressure cryoscanning electron microscopy as described (28). Cells were viewed with a Hitachi S900 scanning electron microscope operated at 2 kV, on a Gatan cryostage. Irregular-sized and -shaped cellular protrusions that can be seen are inferred to be budding sections of the cell. (B) SEM micrograph (bar, 5 μm) of a microcolony of fer1 cells within a dissolution pit on a pyrite surface. Samples were prepared and examined as described (29). When grown with sulfide minerals (pyrite, marcasite, and arsenopyrite) as the energy source, fer1 cells preferentially grow attached to the mineral surface relative to growth in free suspension. (C) TEM image of fer1 (bar, 150 nm), and (D) higher magnification (bar, 25 nm) of the area shown in (C) with arrows, illustrating the single, peripheral membrane surrounding the cell that gives rise to the irregular, pleomorphic morphology (A and B). Samples were prepared for TEM as described (12) and viewed with a Zeiss 10CA at 60 kV.

Although fer1 is phylogenetically similar to F. acidiphilum(Fig. 1), it is physiologically distinct. Both fer1 and F. acidiphilum require yeast extract for growth, but fer1 is also able to grow heterotrophically on yeast extract as the sole energy source (14), whereas F. acidiphilumcannot (12). Fer1 is able to grow between pH 0 and 2.5, with a growth optimum at pH 1.2 (14). In contrast,F. acidiphilum grows over a more restricted pH range, has a higher pH optimum [optimum 1.7; range, 1.3 to 2.2 (12)], and doubles at about one-third the rate at optimal pH [∼32 hours (12)]. Hence, we suggest a new species name,acidarmanus for fer1, within the genusFerroplasma.

In order to determine the proportion of F. acidarmanus at Iron Mountain, we collected sediment and slime samples from the Richmond 5-way in November 1998 for analysis using FISH (13). We collected samples for FISH from the same localities collected from in July 1997 for enrichment cultures (above). In the new samples, 85 ± 7% of the total population in a biofilm (Fig. 3) associated with the sediments hybridized with the Ferroplasma genus–specific probe FER656. The remainder of this biofilm was composed of eukaryotic filaments; hence, essentially the entire prokaryote population wasF. acidarmanus. This species is thus likely to be the Archaeon that was found in high abundance in sediment samples (8). Further analysis during 1999 confirmed that while the microbial diversity varies spatially and seasonally,Ferroplasma species remained a persistent and often dominant microbial constituent at the Richmond 5-way (15).

Figure 3

Examples of the type of biofilm material used to determine the proportion of F. acidarmanus. (A) Stream at B-drift (∼1 m across). The biofilm is anchored within pyritic sediments and forms dense slime streamers in most subsurface (tunnel) waters during the summer and fall months at the Richmond 5-way. (B) DAPI (4′,6′-diamidino-2-phenylindole)–stained microbial cells from B-drift slime streamers shown in (A). Bar (B and C), 10 μm. (C) Cells hybridized with the FER656 probe, from the same field of view as (B). To determine the proportion of cells that hybridized with FER656, counts were made for hybridized cells (13) versus cells stained with DAPI, (30) for four fields of view (0.04 mm2) per well; counts were made for four separate wells.

Microbial populations at subsurface AMD sites are typically poorly studied relative to run-off waters (7) because of the greater difficulty associated with sampling disused and hazardous underground regions. Two other recent studies have identified closely related Thermoplasmales that thrive in similar conditions. One is the previously mentioned F. acidiphilum isolate, obtained from a semi-industrial bioleaching reactor in Eastern Europe [Kazakhstan (12)]. In the other study (Chile; no isolate was obtained), Thermoplasmales were the predominate phylotype in a bioleaching reactor when it was operated under high-sulfate (low-pH) conditions (16). The high metal and sulfate conditions under which some bioleaching reactors are operated may be more representative of highly concentrated subsurface AMD sites than the diluted run-off waters that are more commonly studied. Highly concentrated, acidic solutions clearly enrich for these Archaea, both in the environment and in some bioleaching reactors, suggesting that they may be prevalent at subsurface sites. It is unclear how these closely related Archaea that readily lyse in neutral- pH solutions (12, 14) come to be globally distributed. Current theories for distributing microorganisms would not apply to obligate acidophiles such as these (17).

How microorganisms are able to withstand extremely low pH is still uncertain. Membrane characteristics are believed to be critical features that allow Archaea to thrive in many hostile environments (18). It is notable that at Iron Mountain, where the lowest naturally occurring pH conditions have been reported to date (9), the Archaea that thrive lack cell walls entirely (12) (Fig 2). This suggests that the cytoplasmic membrane composition and construction are key factors for extreme acid tolerance. Only two other organisms, both Thermoplasmaleswithin the genus Picrophilus, are capable of growth at pH 0 (19, 20), the lowest pH recorded to support life. However, their abundance in the environment from which they were cultured is yet unknown.

Acidophily is a ubiquitous trait among the Thermoplasmales, yet they are not known to be important contributors to AMD production. Results of this work suggest that although extremophiles are often confined to sites of small geographic extent (such as hot springs and hydrothermal vents), the physiological function and environmental abundance of these Archaea indicate that they may substantially impact global cycling of iron and sulfur.

  • * To whom correspondence should be addressed at Woods Hole Oceanographic Institute, Department of Marine Chemistry and Geochemistry, Woods Hole, MA 02543, USA. E-mail: kedwards{at}whoi.edu

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